Heart failure from irreversible cardiac tissue damage is a leading cause of disability and mortality in US. Because the heart has negligible intrinsic capacity to regenerate new tissues to replace those lost to injury, there is currently no definitive heart failure treatment, other than organ transplantation. Recent studies have introduced the prospect of replacing damaged heart tissues with healthy cardiomyocytes derived from exogenous pluripotent stem cells or endogenous cardiovascular progenitors. Particularly promising for therapeutic use are embryonic stem (ES) cells and autologous induced pluripotent stem (iPS) cells, which have been definitively shown to be capable of differentiating into cardiomyocytes. However, realizing the full therapeutic potential of stem cells faces numerous hurdles, including the potential for tumor formation, a low rate of cardiomyocyte formation, and an inadequate understanding of how and which progenitors become cardiomyocytes. Current efforts are also hampered by a lack of pharmaceutical agents to boost therapeutic effects of stem cells. Dorsomorphin, the first known small molecule BMP inhibitor, and its structural analogs are among the most potent chemical inducers of in vitro cardiomyogenesis in mouse embryonic stem cells. However, how the BMP inhibitors induce cardiac formation in ES cells is unclear. Curiously, the cardiomyocyte induction occurs in the absence of a significant increase in mesoderm marker expression. As dorsomorphin also has significant activity against the Flk-1 receptor kinase, a key mesoderm maker, the apparent paradox could be due to this off-target effect. To test this and to identify compounds having the highest capacity for cardiac induction, the effects of dorsomorphin on mesoderm and cardiomyocyte formation will be compared to those of a pure BMP inhibitor DMH1, which has no off-target activity against Flk1+ (Aim 1). Treatment with the BMP inhibitors during the first 24 hours of ES cell differentiation is sufficient for robust cardiac induction, and the cardiac induction coincides with a significant reduction in all of the noncardiac mesoderm lineages. Such an inverse relationship suggests that BMP inhibition during the initial stages of ES cell differentiation commits an early common progenitor toward the cardiac-specific developmental program. To test this, the impact of DMH1 on the developmental potential of mesoderm progenitor cells will be examined (Aim 2). Molecular profiling of the DMH1 treated mesoderm cells will also be performed to characterize the early cardiac progenitor cells induced by DMH1. The Aim 3 will take the next logical step to test whether small molecule-based methods found to robustly induce cardiomyocyte formation in vitro can have a beneficial impact on stem cell therapies to improve cardiac remodeling and function in a mouse model of myocardial injury. In conclusion, the proposed study will utilize a class of potent chemical inducers of cardiomyogenesis to elucidate the mechanism of cardiac specification and differentiation, and thereby inform future stem cell-based strategies to treat heart disease.